Fatty acids are major carbon and energy stores in the seeds of many agriculturally important species. For the plant they are essential reserves that support germination and seedling establishment until the plant can manufacture its own building blocks and energy through photosynthesis. Recent published data shows that blocking the mobilisation of these fatty acids prevents or severely compromises establishment (Hayashi et al., 1998, Germain et al., 2001). Clearly, efficient germination and seedling establishment are of vital importance to farmers. In addition, the fatty acids deposited in seeds renders them important foodstuffs for humans and animals. There is considerable interest in modifying the levels and composition of fatty acids in seeds to improve their nutritional quality and health benefit. Furthermore, there is considerable interest in engineering crop plants to produce novel fatty acids with industrial benefit. These can be used as feedstocks for the chemical and healthcare industries, with the aim of reducing reliance on petrochemical feedstocks, it is therefore desirable to produce these molecules in a more ecologically friendly and sustainable way and to develop non-food crops for European agriculture.
Plants that are engineered to produce altered fatty acids rarely produce economically viable levels in seeds. The reasons for this are probably complex but one factor may be their turnover, i.e. some proportion is broken down as they are made. Furthermore, if high levels can be achieved this may compromise the ability of these plants to germinate and establish if these altered fatty acids cannot be used efficiently. Clearly this is detrimental to the commercialisation of these plants.
Fatty acids in seeds are stored in oil bodies in the form of triacylglycerols (3 fatty acid molecules joined to a glycerol backbone) which are laid down during seed development. During germination free fatty acids are released by the action of lipases and the fatty acids enter the β-oxidation pathway which is housed within a specialised organelle the glyoxysome. The fatty acids are then metabolised to produce energy and building blocks for the cell. Control of biochemical pathways frequently resides near the beginning of the pathway and in several cases transport steps have been shown to exert high flux control coefficients. This means that transporting a molecule, for example, from compartment A to compartment B is Often an important step in determining the overall rate of the pathway.
There is some evidence to suggest from human studies that proteins of the ATP Binding Cassette family (referred to as ABCs) are involved in fatty acid transport. ABCs are integral membrane proteins that transport a wide variety of molecules across membranes. It is known from the prior art that X-linked adrenoleukodystrophy (X-ALD) is associated with a particular gene mutation (Moser et al., 1993). The clinical symptoms of the disease, results in increasing neurological impairment, progressive mental and physical disability, and eventually death in late childhood or early teens. Biochemically these patients fail to break down very long chain fatty acids. The gene mutated in X-ALD is an ABC transporter closely related to but not identical to another mammalian peroxisomal ABC transporter, PMP70. It is now known that there are 4 of these peroxisomal ABC genes in humans (PMP70, PMP70R, ALD and ALDR. In addition, two homologous genes have been identified in yeast S. cerevisiae (PXA1 and PXA2 also known as PAT1 and PAT2) and have been shown to be transporters of fatty acyl CoAs (Hettema et al., 1996). PXA2 is also known as the COMATOSE (CTS) gene (Russell et al., 2000; Footitt et al., 2002).
Glyoxysomes are cytoplasmic organelles unique to plants. Glyoxysomes are a specialised form of peroxisomes but they also contain enzymes of the glyoxalate cycle. They are abundantly present in the endosperm or cotyledons of oil-rich seeds. It is not known how fatty acids are transported into glyoxysomes.
It is therefore desirous to identify a transport protein or a regulator protein that is involved with the rate of entry of fatty acids into the degradation pathway. Identification and characterisation of the proteins that transport fatty acids into glyoxysomes would offer an attractive target for biotechnology with a view to repress or promote growth or to alter the spectrum of fatty acids which can be utilised by the plant.